Direct binding of soluble HLA-G to CD158d has not been demonstrated. We performed random mutagenesis of the extracellular domain of CD158d and used a reliable HLA-G interaction assay and microscopy to screen for mutants with specific defects. The assay consists of incubating CD158d+ cells with soluble HLA-G and performing confocal microscopy to monitor accumulation of HLA-G in endosomes where CD158d resides. CD158d is a constitutive resident of early endosomes. Mutants with single amino acid changes were examined by this HLA-G endosomal loading assay. During incubation with cells expressing wild-type CD158d, soluble HLA-G accumulates gradually over time in CD158d+ endosomes. Three categories of mutants were identified: those with a defect in HLA-G binding (CD158d in endosomes, no HLA-G), those with a defect in trafficking to endosomes (CD158d at the plasma membrane) and those with a double defect (CD158d at the plasma membrane, no HLA-G). The results demonstrated that the folding and structure of the CD158d extracellular domain controls receptor internalization into endosomes and HLA-G binding, and that these two properties can be separated.